I have set up a PCR using primers that amplify only the exons of my gene of interest. The template I am using is cDNA (mouse samples). I set up this PCR a year ago with an annealing temperature of 57 C, using Kapa Master Mix (the product size is about 2.8 kb). I repeated this PCR 3 weeks ago and I could get sharp bands for some of the samples (Fig. 1), but when I repeated the PCR again (2 days after), I only got smears (Fig. 2) (In the next repeatations changing extention and cycle numbers, sometimes for some samples bands appeared (Fig 3) and in the next round for the same samples I only got smears!) I changed each component and used filtered tips. I also tried different extension times and reduced cycles. I have used diluted templates (1/2, 1/4 and 1/8). I cannot understand why the PCR that worked is not working anymore! I have changed the stock of primers and I still get smear in my samples and water control (Fig 4). When I use other primers targeting different gene, it works (Fig 2). Regarding the water control, using the same master mix but different primers resulted in a clean water control compared to the water control for my gene of interest.
Could you please share your thoughts on this? I appreciate any comments.
you have post pcr contamination of a reagent/working area/plasticware. It amplifies very well so the expected band self anneals and forms long smears of over amplified product. The contamination comes from one set of primers originally so no other primer set will anneal or amplify this contamination so these other primer sets are not contaminated.
You are going to have to change all reagents and clean all of your pipettes. Move to another working area and use new primers and other researchers pcr reagents and plasticware or design new primers with one primer outside of the current pcr product ( make a larger product) then the new primers cannot anneal to the original contamination
Paul Rutland Thank you for your reply. I have changed everything including pipettes, reagents and working bench! As I said I reordered the same primers and reconstituted them (in order to exclude the possibility of contamination of the primers) and used them but again I had smear. I think for the next step, I will take your suggestion regarding the design of the new primers.
Paul Rutland Regarding the smear in my negative control, I found this page
https://international.neb.com/faqs/0001/01/01/what-causes-an-occasional-smear-in-a-negative-control-with-no-template-present
that they mentioned duo to the low Km values of the DNA polymerases for DNA, we might have smear in the negative control. I don't know how much sense this makes, but if it is the right explanation, I can't understand why I have smear in my test samples. I was thinking of reducing the concentration of my primers before designing a new one. I would be grateful if you could let me know what you think about this.
Thank you for that Atefeh Sadeghi . I did not know about this effect with proof reading polymerases. Your problem looks different to me in that you not only have a smear in the NTC but also you are not getting a band in some samples and your smear does look like a high molecular weight smear looking more like concatenated product and I cannot see that it is a single primer binding issue when new primers still have a problem Is the ntc amplification only occasional or is it now a reproducible result?, Also other primer pairs are working well and presumably their NTCs are without smears even with the same enzyme. If you do design new primers then I would suggest having a pre and post pcr area and no transfer of material between them if possible. So open the tubes away from the pcr setup area ( maybe in the gel running area) and use a pipette that does not come back to your setup area and discard any gloves that have touched gels or buffer before returning to the setup area
Paul Rutland Thank you, it is an occasional problem, as you can see in figure 1 and 3 I did not have the problem and in figure 2 and 4 I did. I will consider your advice and thank you for your help.
It might be that your primer concentration is very high. If your negative control is good, the effect might come from old or degraded samples.
hi Atefeh Sadeghi, you may have a couple of things that come together.
1) Put the gel comb into hot water for a few minutes and clean well with paper. What can happened is that a thin film of gel dries on the comb and is left behind in the next gel. This film retains DNA from migration and tends to make smears.
2) too high primer concentration and artifact amplification will happen. As you mention reducing their concentration should mostly solve that problem.
3) If other people in the lab use the same reagents make your own aliquot. Specially EB (elution buffer) and water.
4) Gloves are useful to protect yourself. Rinse hand often. Clean area with water simple tap water. Separate template (RNA cDNA) from the area you prepare the mastermix with the primers.
5) If you have a PCR product contamination you would get a sharp band in mostly every reaction.
6) design primers not too GC rich at the 3' end as it soon or later will start on a GC rich region of your template.
7) cDNA after RT maybe in acidic solution and DNA gets hydrolyzed under such condition. Just buffer cDNA with 10mM Tris pH9.
Hope this may help.
Hannes
Getaw Deresse Thank you, I am also thinking about the primer concentration and will try again with less primer.
Hannes Richter Thanks for the detailed comment, I repeated the PCR yesterday and put everything (including the comb) under UV and even did the PCR under the hood and in the result the DW was clean but I still have smear in the test wells. As you said I have couple of things come together! So today I will repeat the PCR with a lower concentration of primer and if it does not work again I will order a new primer considering your point.
UV is not such a good idea. I used it to cross link DNA or RNA to membranes for Southern or Northern blot. With UV you just fix the DNA to the object it is on but do not remove it.
DNA is well soluble in water but not in EtOH.
The smear is odd but it seems to be due to the PCR.
Could you do a reaction including everything except the primers.
Have you changed the mastermix ?
How much RNA + cDNA do you add in the PCR reaction.
Have you changed thermocycler ?
WOW, I didn't know that about water! But it makes sense. Today I took your advice and put the comb in warm water and cleaned it afterwards. This is a good idea, my PCR with half concentration of primers is running now and I will tell you the result. If it does not work, I will do a PCR without primers. The master mix is from a new batch and a new aliquot.
I use cDNA (100 ng, 8 µl), but I have also diluted this 1/2, 1/4 and 1/8 and still the result was a smear!
I changed the thermocycler once (I did it in another lab with their equipment), but I still had a smear!
Thank you for following the question and helping me.
Your template (cDNA) quality may have been altered. Did you check the quality of the template, were they good enough?
Sumaiya Binte Hannan Thanks for the answer. I used the same cDNA with a different primer and it worked, and also for the same primer I have a strong band for some samples and smear or weak for some others. So I think the cDNA is ok. Also once for another sample (same method of RNA extraction and cDNA synthesis) I used Tapestation and the quality of my sample was good.
Manal Al-nasrawi Thank you very much. I changed my primers sequence to one with a higher annealing temperature and it worked.
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